Tools for Enhancement of Diagnostic Immunoassay Applications

Tim M. Jentz, Wendy D. Nelson, Ph.D., Gary Opperman

SurModics, Inc., Eden Prairie, MN

Contact InfoTim M Jentz

tjentz@surmodics.com

AbstractOptimization of immunoassay applications is often hampered by issues such as non-specific binding, matrix interferences, destabilization of antibody/antigen interactions, and limited sensitivity. SurModics offers tools to address these problems and provides the framework to developsensitive, reproducible, and robust immunoassays. Stabilization of antibody-protein interactions is fundamental for achieving a robust and reproducible assay. Novel stabilizer formulations have been developed to provide protein stability in both the dried state as well as in solution. Thedata presented here show improved stability compared to both common in-house formulations as well as other competitors’ solutions. Problems with non-specific binding and matrix interferences can cause high background, loss of sensitivity, and false positives in many diagnosticapplications. These issues were mitigated by use of protein-free blocking solutions or novel passivating surface chemistry for microarray applications. Data here suggest the TRIDIA® surface chemistry provides significantly reduced non-specific binding in protein arrays. Shown here aredata from a new, protein-free diluent designed to reduce matrix interferences and to be substituted with ease into your existing assay protocol. Whether it is in solution or on a surface, SurModics provides the tools necessary to develop and optimize immunoassays that are sensitive,reproducible, and robust.

Microarray Applications

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Figure #1: Dried antibody stability with StabilCoat® and StabilGuard® stabilizers

Methods: A capture antibody was stabilized with different commercially available stabilizers.This accelerated stability study challenged the captured antibody at 37ºC. The retainedactivity of the captured antibody was evaluated in a sandwich ELISA over one year bycomparing the immunoassay signal produced by the 4ºC control versus 37ºC. Retainedactivity values are determined by dividing the average optical density at 37ºC by the averageoptical density at 4ºC, then multiplying by 100.

Results: At the one-year stability time point, StabilCoat and StabilGuard stabilizersdemonstrated greater than 90% retained activity. The sustained functional activity suggestsSurModics’ stabilizers were able to preserve the functional conformation of the driedantibody.

Figure #2: In-house stabilizer comparison

Methods: Using the ELISA methods described in Figure #1, StabilCoat and StabilGuardstabilizers were compared versus common in-house stabilizers.

Results: After one week at 37ºC storage, StabilCoat and StabilGuard stabilizersdemonstrated nearly 100% retained activity.

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Figure #3: Troponin In-Solution Stability

Methods: Native Human Cardiac Troponin-I was diluted to 10 µg/mL in a MOPS buffer, acommercially available in-solution competitor and SurModics stabilizer in development.The stabilized Troponin was tested in a Troponin sandwich ELISA over time andcompared to a freshly prepared control.

Results: SurModics in-development stabilizer demonstrated ~ 95% retained activitycompared to the freshly prepared control after 154 days at 4ºC.

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TroponinTroponin(ng/mL)(ng/mL)

TRIDIA EpoxideSurface Epoxysilane A Epoxysilane B

Figure #7: Analysis of Troponin on a Protein Microarray

Methods: Monoclonal antibody to Troponin I (AbCam) at 1 mg/mL was printedon all slides and incubated overnight. Slides were assembled into 16 well platesusing ProPlate® slide chambers (Grace Bio-Labs) and then washed/blocked inPBS-Tween (0.05%) (PBS-T) containing 5% BSA. Troponin I was diluted,incubated for 1.5 hours and washed. Primary antibody (Rb x hTroponin; 1

g/mL) and secondary (Gt x RbIgG-Cy3; 1 g/mL) were used to detect TroponinI. Slides were washed, centrifuged dry and then scanned on an Axon 4200ALscanner in the 532 nm channel.

Results: The TRIDIA surface blocked non-specific binding compared tocommercially available epoxysilane surfaces. The epoxysilane slides show non-specific binding at the higher Troponin levels. The TRIDIA slides display moredistinct spots with consistently low background across the slide.

Figure #9: Western Blot of Recombinant mIL-2

Methods: Recombinant mIL-2 protein (R&D Systems, Inc) dilutionswere prepared, run on a gradient gel and transferred to a nitrocellulosemembrane. StabilBlot™ buffer was used as both the blocker andantibody diluent. Primary antibody (Gt x mIL-2; 0.5ug/mL) (R&DSystems, Inc) and secondary antibody (DK x GtIgG-HRP; 0.05ug/mL)(GenScript) were used to detect mIL-2. The membrane was developedwith SurModics’ BioFX® CHMM HRP substrate and captured using aCCD camera.

Results: StabilBlot buffer enhanced the signal and detection limit forrmIL-2 protein over a competitor and several common in-house bufferformulations. Since StabilBlot buffer is synthetic, cross-reactivityissues, such as those observed here for BSA, are eliminated.

Summary• Superior protein stability/activity in both dried and in-solution formats• Decreased HAMA false positives and demonstrated minimal dynamic range reduction• Enhanced signal to noise ratio in assays with matrix interferences• Improved signal and eliminated cross-reactivity concerns on membranes• TRIDIA surface chemistry provides:

– Support for cell array formats– Superior epoxy surface– Simultaneous biomolecule immobilization and passivation

Demonstrated Improved Assay Performance Across Multiple Diagnostic Applications

StabilBlot is a trademark of SurModics, Inc.

StabilGuard, StabilCoat, and TRIDIA are registered trademarks of SurModics, Inc.

CodeLink is a trademark of a GE Healthcare Company

ProPlate is a registered trademark of Grace Bio-Labs, Inc.

Matrix Interference Reduction

Troponin In-Solution Stability

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MOPS buffer

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Figure #6: Signal to Noise Ratio

Methods:

Signal to Noise Ratio calculation: S/N = Top standard ODFalse positive OD

Results: SurModics Assay Diluent demonstrates superior signal to noise compared to PBS andthe competitor diluents.

Figure #5: Retains Dynamic Range

Methods: A Troponin standard curve was prepared in PBS, the SurModics Assay Diluent, andfour competitors.

2nd point of std curve OD– 5th point of std curve OD

= Dynamic Range

Results: Figure #5 demonstrates a minimal effect on dynamic range when SurModics AssayDiluent was used to prepare a Troponin standard curve compared to other competitors.

Figure #4: False Positive Reduction

Methods: Serum containing human anti-mouse antibody (HAMA) produced a false positive in a Troponin sandwichELISA. The false positive results occurred when HAMA caused bridging between the mouse monoclonal captureantibody and the mouse monoclonal detection antibody. The results shown in Figure #4 were obtained by addingserum containing elevated HAMA levels diluted with either PBS, SurModics Assay Diluent, or four other competitors.

Results: Figure #4 demonstrates ~57% blocking of the false positive signal using the SurModics Assay Diluent.

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HAMA False Positive

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